The Asp99–Arg188 salt bridge of the Pseudomonas aeruginosa HemO is critical in allowing conformational flexibility during catalysis

Geoffrey A. Heinzl, Weiliang Huang, Elizabeth Robinson, Fengtian Xue, Pierre Moenne-Loccoz, Angela Wilks

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Abstract: The P. aeruginosa iron-regulated heme oxygenase (HemO) is required within the host for the utilization of heme as an iron source. As iron is essential for survival and virulence, HemO represents a novel antimicrobial target. We recently characterized small molecule inhibitors that bind to an allosteric site distant from the heme pocket, and further proposed binding at this site disrupts a nearby salt bridge between D99 and R188. Herein, through a combination of site-directed mutagenesis and hydrogen–deuterium exchange mass spectrometry (HDX-MS), we determined that the disruption of the D99–R188 salt bridge leads to significant decrease in conformational flexibility within the distal and proximal helices that form the heme-binding site. The RR spectra of the resting state Fe(III) and reduced Fe(II)-deoxy heme-HemO D99A, R188A and D99/R188A complexes are virtually identical to those of wild-type HemO, indicating no significant change in the heme environment. Furthermore, mutation of D99 or R188 leads to a modest decrease in the stability of the Fe(II)-O2 heme complex. Despite this slight difference in Fe(II)-O2 stability, we observe complete loss of enzymatic activity. We conclude the loss of activity is a result of decreased conformational flexibility in helices previously shown to be critical in accommodating variation in the distal ligand and the resulting chemical intermediates generated during catalysis. Furthermore, this newly identified allosteric binding site on HemO represents a novel alternative drug-design strategy to that of competitive inhibition at the active site or via direct coordination of ligands to the heme iron.

Original languageEnglish (US)
Pages (from-to)1057-1070
Number of pages14
JournalJournal of Biological Inorganic Chemistry
Volume23
Issue number7
DOIs
StatePublished - Oct 1 2018

Fingerprint

Heme Oxygenase (Decyclizing)
Catalysis
Heme
Pseudomonas aeruginosa
Salts
Iron
Allosteric Site
Binding Sites
Ligands
Mutagenesis
Drug Design
Site-Directed Mutagenesis
Mass spectrometry
Virulence
Mass Spectrometry
Catalytic Domain
Mutation
Molecules
Pharmaceutical Preparations

Keywords

  • Biliverdin
  • Heme oxygenase
  • Oxygen activation
  • Protein dynamics
  • Pseudomonas aeruginosa

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry

Cite this

The Asp99–Arg188 salt bridge of the Pseudomonas aeruginosa HemO is critical in allowing conformational flexibility during catalysis. / Heinzl, Geoffrey A.; Huang, Weiliang; Robinson, Elizabeth; Xue, Fengtian; Moenne-Loccoz, Pierre; Wilks, Angela.

In: Journal of Biological Inorganic Chemistry, Vol. 23, No. 7, 01.10.2018, p. 1057-1070.

Research output: Contribution to journalArticle

Heinzl, Geoffrey A. ; Huang, Weiliang ; Robinson, Elizabeth ; Xue, Fengtian ; Moenne-Loccoz, Pierre ; Wilks, Angela. / The Asp99–Arg188 salt bridge of the Pseudomonas aeruginosa HemO is critical in allowing conformational flexibility during catalysis. In: Journal of Biological Inorganic Chemistry. 2018 ; Vol. 23, No. 7. pp. 1057-1070.
@article{cbac1fd03ce44d04a2970001d5508bd3,
title = "The Asp99–Arg188 salt bridge of the Pseudomonas aeruginosa HemO is critical in allowing conformational flexibility during catalysis",
abstract = "Abstract: The P. aeruginosa iron-regulated heme oxygenase (HemO) is required within the host for the utilization of heme as an iron source. As iron is essential for survival and virulence, HemO represents a novel antimicrobial target. We recently characterized small molecule inhibitors that bind to an allosteric site distant from the heme pocket, and further proposed binding at this site disrupts a nearby salt bridge between D99 and R188. Herein, through a combination of site-directed mutagenesis and hydrogen–deuterium exchange mass spectrometry (HDX-MS), we determined that the disruption of the D99–R188 salt bridge leads to significant decrease in conformational flexibility within the distal and proximal helices that form the heme-binding site. The RR spectra of the resting state Fe(III) and reduced Fe(II)-deoxy heme-HemO D99A, R188A and D99/R188A complexes are virtually identical to those of wild-type HemO, indicating no significant change in the heme environment. Furthermore, mutation of D99 or R188 leads to a modest decrease in the stability of the Fe(II)-O2 heme complex. Despite this slight difference in Fe(II)-O2 stability, we observe complete loss of enzymatic activity. We conclude the loss of activity is a result of decreased conformational flexibility in helices previously shown to be critical in accommodating variation in the distal ligand and the resulting chemical intermediates generated during catalysis. Furthermore, this newly identified allosteric binding site on HemO represents a novel alternative drug-design strategy to that of competitive inhibition at the active site or via direct coordination of ligands to the heme iron.",
keywords = "Biliverdin, Heme oxygenase, Oxygen activation, Protein dynamics, Pseudomonas aeruginosa",
author = "Heinzl, {Geoffrey A.} and Weiliang Huang and Elizabeth Robinson and Fengtian Xue and Pierre Moenne-Loccoz and Angela Wilks",
year = "2018",
month = "10",
day = "1",
doi = "10.1007/s00775-018-1609-x",
language = "English (US)",
volume = "23",
pages = "1057--1070",
journal = "Journal of Biological Inorganic Chemistry",
issn = "0949-8257",
publisher = "Springer Verlag",
number = "7",

}

TY - JOUR

T1 - The Asp99–Arg188 salt bridge of the Pseudomonas aeruginosa HemO is critical in allowing conformational flexibility during catalysis

AU - Heinzl, Geoffrey A.

AU - Huang, Weiliang

AU - Robinson, Elizabeth

AU - Xue, Fengtian

AU - Moenne-Loccoz, Pierre

AU - Wilks, Angela

PY - 2018/10/1

Y1 - 2018/10/1

N2 - Abstract: The P. aeruginosa iron-regulated heme oxygenase (HemO) is required within the host for the utilization of heme as an iron source. As iron is essential for survival and virulence, HemO represents a novel antimicrobial target. We recently characterized small molecule inhibitors that bind to an allosteric site distant from the heme pocket, and further proposed binding at this site disrupts a nearby salt bridge between D99 and R188. Herein, through a combination of site-directed mutagenesis and hydrogen–deuterium exchange mass spectrometry (HDX-MS), we determined that the disruption of the D99–R188 salt bridge leads to significant decrease in conformational flexibility within the distal and proximal helices that form the heme-binding site. The RR spectra of the resting state Fe(III) and reduced Fe(II)-deoxy heme-HemO D99A, R188A and D99/R188A complexes are virtually identical to those of wild-type HemO, indicating no significant change in the heme environment. Furthermore, mutation of D99 or R188 leads to a modest decrease in the stability of the Fe(II)-O2 heme complex. Despite this slight difference in Fe(II)-O2 stability, we observe complete loss of enzymatic activity. We conclude the loss of activity is a result of decreased conformational flexibility in helices previously shown to be critical in accommodating variation in the distal ligand and the resulting chemical intermediates generated during catalysis. Furthermore, this newly identified allosteric binding site on HemO represents a novel alternative drug-design strategy to that of competitive inhibition at the active site or via direct coordination of ligands to the heme iron.

AB - Abstract: The P. aeruginosa iron-regulated heme oxygenase (HemO) is required within the host for the utilization of heme as an iron source. As iron is essential for survival and virulence, HemO represents a novel antimicrobial target. We recently characterized small molecule inhibitors that bind to an allosteric site distant from the heme pocket, and further proposed binding at this site disrupts a nearby salt bridge between D99 and R188. Herein, through a combination of site-directed mutagenesis and hydrogen–deuterium exchange mass spectrometry (HDX-MS), we determined that the disruption of the D99–R188 salt bridge leads to significant decrease in conformational flexibility within the distal and proximal helices that form the heme-binding site. The RR spectra of the resting state Fe(III) and reduced Fe(II)-deoxy heme-HemO D99A, R188A and D99/R188A complexes are virtually identical to those of wild-type HemO, indicating no significant change in the heme environment. Furthermore, mutation of D99 or R188 leads to a modest decrease in the stability of the Fe(II)-O2 heme complex. Despite this slight difference in Fe(II)-O2 stability, we observe complete loss of enzymatic activity. We conclude the loss of activity is a result of decreased conformational flexibility in helices previously shown to be critical in accommodating variation in the distal ligand and the resulting chemical intermediates generated during catalysis. Furthermore, this newly identified allosteric binding site on HemO represents a novel alternative drug-design strategy to that of competitive inhibition at the active site or via direct coordination of ligands to the heme iron.

KW - Biliverdin

KW - Heme oxygenase

KW - Oxygen activation

KW - Protein dynamics

KW - Pseudomonas aeruginosa

UR - http://www.scopus.com/inward/record.url?scp=85052966478&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85052966478&partnerID=8YFLogxK

U2 - 10.1007/s00775-018-1609-x

DO - 10.1007/s00775-018-1609-x

M3 - Article

C2 - 30194537

AN - SCOPUS:85052966478

VL - 23

SP - 1057

EP - 1070

JO - Journal of Biological Inorganic Chemistry

JF - Journal of Biological Inorganic Chemistry

SN - 0949-8257

IS - 7

ER -